AXMI-205 pesticidal gene and method for its use

ABSTRACT

Compositions and methods for conferring pesticidal activity to bacteria, plants, plant cells, tissues and seeds are provided. Compositions comprising a coding sequence for pesticidal polypeptides are provided. The coding sequences can be used in DNA constructs or expression cassettes for transformation and expression in plants and bacteria. Compositions also comprise transformed bacteria, plants, plant cells, tissues, and seeds. In particular, isolated pesticidal nucleic acid molecules are provided. Additionally, amino acid sequences corresponding to the polynucleotides are encompassed. In particular, the present invention provides for nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequence shown in SEQ ID NO:2, 3, or 4, the nucleotide sequence set forth in SEQ ID NO:1, 9, 10, or 11, as well as variants and fragments thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/828,594, filed Jul. 1, 2010, which claims the benefit of U.S.Provisional Application Ser. No. 61/222,778, filed Jul. 2, 2009, thecontents of which are herein incorporated by reference in theirentirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named“APA066US01DDSeqList,” created on Oct. 12, 2013, and having a size of 63kilobytes and is filed concurrently with the specification. The sequencelisting contained in this ASCII formatted document is part of thespecification and is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to the field of molecular biology. Provided arenovel genes that encode pesticidal proteins. These proteins and thenucleic acid sequences that encode them are useful in preparingpesticidal formulations and in the production of transgenicpest-resistant plants.

BACKGROUND OF THE INVENTION

Introduction of DDT (dichloro-diphenyl-trichloroethane) and thefollowing move towards indiscriminate use of synthetic chemicalinsecticides led to the contamination of water and food sources,poisoning of non-target beneficial insects and development of insectpests resistant to the chemical insecticides. Increased public concernsabout the adverse environmental effects of indiscriminate use ofchemical insecticides prompted a search for alternative methods forinsect pest control.

One of the promising alternatives has been the use of biological controlagents. There is well-documented history of safe application of Bt (B.thuringiensis, a gram positive soil bacterium) as effectivebiopesticides and a number of reports of expression of delta-endotoxingene(s) in crop plants are available. Only a few insecticidal sprays arerequired on Bt transgenic crops, which not only save cost and time, butalso reduce health risks. In some cases, insects can develop resistanceto different insecticidal compounds, which raises the need to identifyalternative biological control agents for pest control.

SUMMARY OF INVENTION

Compositions and methods for conferring pesticidal activity to bacteria,plants, plant cells, tissues and seeds are provided. Compositionsinclude nucleic acid molecules encoding sequences for pesticidal andinsecticidal polypeptides, vectors comprising those nucleic acidmolecules, and host cells comprising the vectors. Compositions alsoinclude the pesticidal polypeptide sequences and antibodies to thosepolypeptides. The nucleotide sequences can be used in DNA constructs orexpression cassettes for transformation and expression in organisms,including microorganisms and plants. The nucleotide or amino acidsequences may be synthetic sequences that have been designed forexpression in an organism including, but not limited to, a microorganismor a plant. Compositions also comprise transformed bacteria, plants,plant cells, tissues, and seeds.

In particular, isolated or recombinant nucleic acid molecules areprovided that encode a pesticidal protein. Additionally, amino acidsequences corresponding to the pesticidal protein are encompassed. Inparticular, the present invention provides for an isolated nucleic acidmolecule comprising a nucleotide sequence encoding the amino acidsequence shown in SEQ ID NO:2, 3, 4, 5, 6, 7, or 8 or a nucleotidesequence set forth in SEQ ID NO:1, 9, 10, or 11, as well as variants andfragments thereof. Nucleotide sequences that are complementary to anucleotide sequence of the invention, or that hybridize to a sequence ofthe invention are also encompassed.

Methods are provided for producing the polypeptides of the invention,and for using those polypeptides for controlling or killing alepidopteran, coleopteran, nematode, or dipteran pest. Methods and kitsfor detecting the nucleic acids and polypeptides of the invention in asample are also included.

The compositions and methods of the invention are useful for theproduction of organisms with enhanced pest resistance or tolerance.These organisms and compositions comprising the organisms are desirablefor agricultural purposes. The compositions of the invention are alsouseful for generating altered or improved proteins that have pesticidalactivity, or for detecting the presence of pesticidal proteins ornucleic acids in products or organisms.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows an alignment of AXMI-205 (SEQ ID NO:2) with MACPF proteinsfrom Photorhabdus luminescens (SEQ ID NO:14) and Clavibactermichiganensis (SEQ ID NO:15).

DETAILED DESCRIPTION

The present invention is drawn to compositions and methods forregulating pest resistance or tolerance in organisms, particularlyplants or plant cells. By “resistance” is intended that the pest (e.g.,insect) is killed upon ingestion or other contact with the polypeptidesof the invention. By “tolerance” is intended an impairment or reductionin the movement, feeding, reproduction, or other functions of the pest.The methods involve transforming organisms with a nucleotide sequenceencoding a pesticidal protein of the invention. In particular, thenucleotide sequences of the invention are useful for preparing plantsand microorganisms that possess pesticidal activity. Thus, transformedbacteria, plants, plant cells, plant tissues and seeds are provided.Compositions are pesticidal nucleic acids and proteins of bacterialspecies. The sequences find use in the construction of expressionvectors for subsequent transformation into organisms of interest, asprobes for the isolation of other homologous (or partially homologous)genes, and for the generation of altered pesticidal proteins by methodsknown in the art, such as domain swapping or DNA shuffling. The proteinsfind use in controlling or killing lepidopteran, coleopteran, dipteran,and nematode pest populations and for producing compositions withpesticidal activity.

By “pesticidal toxin” or “pesticidal protein” is intended a toxin thathas toxic activity against one or more pests, including, but not limitedto, members of the Lepidoptera, Diptera, and Coleoptera orders, or theNematoda phylum, or a protein that has homology to such a protein.Pesticidal proteins have been isolated from organisms including, forexample, Bacillus sp., Clostridium bifermentans and Paenibacilluspopilliae. Pesticidal proteins include amino acid sequences deduced fromthe full-length nucleotide sequences disclosed herein, and amino acidsequences that are shorter than the full-length sequences, either due tothe use of an alternate downstream start site, or due to processing thatproduces a shorter protein having pesticidal activity. Processing mayoccur in the organism the protein is expressed in, or in the pest afteringestion of the protein.

Thus, provided herein are novel isolated or recombinant nucleotidesequences that confer pesticidal activity. Also provided are the aminoacid sequences of the pesticidal proteins. The protein resulting fromtranslation of this gene allows cells to control or kill pests thatingest it.

Isolated Nucleic Acid Molecules, and Variants and Fragments Thereof

One aspect of the invention pertains to isolated or recombinant nucleicacid molecules comprising nucleotide sequences encoding pesticidalproteins and polypeptides or biologically active portions thereof, aswell as nucleic acid molecules sufficient for use as hybridizationprobes to identify nucleic acid molecules encoding proteins with regionsof sequence homology. As used herein, the term “nucleic acid molecule”is intended to include DNA molecules (e.g., recombinant DNA, cDNA orgenomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA.

An “isolated” nucleic acid sequence (or DNA) is used herein to refer toa nucleic acid sequence (or DNA) that is no longer in its naturalenvironment, for example in an in vitro or in a recombinant bacterial orplant host cell. In some embodiments, an “isolated” nucleic acid is freeof sequences (preferably protein encoding sequences) that naturallyflank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends ofthe nucleic acid) in the genomic DNA of the organism from which thenucleic acid is derived. For purposes of the invention, “isolated” whenused to refer to nucleic acid molecules excludes isolated chromosomes.For example, in various embodiments, the isolated nucleic acid moleculeencoding a pesticidal protein can contain less than about 5 kb, 4 kb, 3kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturallyflank the nucleic acid molecule in genomic DNA of the cell from whichthe nucleic acid is derived. A pesticidal protein that is substantiallyfree of cellular material includes preparations of protein having lessthan about 30%, 20%, 10%, or 5% (by dry weight) of non-pesticidalprotein (also referred to herein as a “contaminating protein”).

Nucleotide sequences encoding the proteins of the present inventioninclude the sequence set forth in SEQ ID NO:1, 9, 10, or 11, andvariants, fragments, and complements thereof. By “complement” isintended a nucleotide sequence that is sufficiently complementary to agiven nucleotide sequence such that it can hybridize to the givennucleotide sequence to thereby form a stable duplex. The correspondingamino acid sequence for the pesticidal protein encoded by thisnucleotide sequence are set forth in SEQ ID NO:2, 3, or 4.

Nucleic acid molecules that are fragments of these nucleotide sequencesencoding pesticidal proteins are also encompassed by the presentinvention. By “fragment” is intended a portion of the nucleotidesequence encoding a pesticidal protein. A fragment of a nucleotidesequence may encode a biologically active portion of a pesticidalprotein, or it may be a fragment that can be used as a hybridizationprobe or PCR primer using methods disclosed below. Nucleic acidmolecules that are fragments of a nucleotide sequence encoding apesticidal protein comprise at least about 50, 100, 200, 300, 400, 500,600, 700, 800, 900, 1000, 1100, 1200, 1300, 1350, 1400, 1450, 1500,1550, 1600 contiguous nucleotides, or up to the number of nucleotidespresent in a full-length nucleotide sequence encoding a pesticidalprotein disclosed herein, depending upon the intended use. By“contiguous” nucleotides is intended nucleotide residues that areimmediately adjacent to one another. Fragments of the nucleotidesequences of the present invention will encode protein fragments thatretain the biological activity of the pesticidal protein and, hence,retain pesticidal activity. By “retains activity” is intended that thefragment will have at least about 30%, at least about 50%, at leastabout 70%, 80%, 90%, 95% or higher of the pesticidal activity of thepesticidal protein. In one embodiment, the pesticidal activity iscoleoptericidal activity. In another embodiment, the pesticidal activityis lepidoptericidal activity. In another embodiment, the pesticidalactivity is nematocidal activity. In another embodiment, the pesticidalactivity is diptericidal activity. Methods for measuring pesticidalactivity are well known in the art. See, for example, Czapla and Lang(1990) J. Econ. Entomol. 83:2480-2485; Andrews et al. (1988) Biochem. J.252:199-206; Marrone et al. (1985) J. of Economic Entomology 78:290-293;and U.S. Pat. No. 5,743,477, all of which are herein incorporated byreference in their entirety.

A fragment of a nucleotide sequence encoding a pesticidal protein thatencodes a biologically active portion of a protein of the invention willencode at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250,300, 350, 400, 450, 500, 550, or 600 contiguous amino acids, or up tothe total number of amino acids present in a full-length pesticidalprotein of the invention. In some embodiments, the fragment is anN-terminal or a C-terminal truncation of at least about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or more aminoacids relative to SEQ ID NO:2, 3, or 4. In some embodiments, thefragments encompassed herein result from the removal of the C-terminal1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25 or more amino acids, e.g., by proteolysis or by insertion of a stopcodon in the coding sequence.

Preferred pesticidal proteins of the present invention are encoded by anucleotide sequence sufficiently identical to the nucleotide sequence ofSEQ ID NO:1, 9, 10, or 11. By “sufficiently identical” is intended anamino acid or nucleotide sequence that has at least about 60% or 65%sequence identity, about 70% or 75% sequence identity, about 80% or 85%sequence identity, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or greater sequence identity compared to a reference sequence usingone of the alignment programs described herein using standardparameters. One of skill in the art will recognize that these values canbe appropriately adjusted to determine corresponding identity ofproteins encoded by two nucleotide sequences by taking into accountcodon degeneracy, amino acid similarity, reading frame positioning, andthe like.

To determine the percent identity of two amino acid sequences or of twonucleic acids, the sequences are aligned for optimal comparisonpurposes. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent identity=number of identical positions/total number of positions(e.g., overlapping positions)×100). In one embodiment, the two sequencesare the same length. In another embodiment, the comparison is across theentirety of the reference sequence (e.g., across the entirety of one ofSEQ ID NO:1, 9, 10, or 11, or across the entirety of one of SEQ ID NO:2,3, 4, 5, 6, 7, or 8). The percent identity between two sequences can bedetermined using techniques similar to those described below, with orwithout allowing gaps. In calculating percent identity, typically exactmatches are counted.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A nonlimiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad.Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTNand BLASTX programs of Altschul et al. (1990) J. Mol. Biol. 215:403.BLAST nucleotide searches can be performed with the BLASTN program,score=100, wordlength=12, to obtain nucleotide sequences homologous topesticidal-like nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the BLASTX program, score=50,wordlength=3, to obtain amino acid sequences homologous to pesticidalprotein molecules of the invention. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-Blast can be used to perform an iterated search thatdetects distant relationships between molecules. See Altschul et al.(1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blastprograms, the default parameters of the respective programs (e.g.,BLASTX and BLASTN) can be used. Alignment may also be performed manuallyby inspection.

Another non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the ClustalW algorithm (Higgins et al.(1994) Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences andaligns the entirety of the amino acid or DNA sequence, and thus canprovide data about the sequence conservation of the entire amino acidsequence. The ClustalW algorithm is used in several commerciallyavailable DNA/amino acid analysis software packages, such as the ALIGNXmodule of the Vector NTI Program Suite (Invitrogen Corporation,Carlsbad, Calif.). After alignment of amino acid sequences withClustalW, the percent amino acid identity can be assessed. Anon-limiting example of a software program useful for analysis ofClustalW alignments is GENEDOC™. GENEDOC™ (Karl Nicholas) allowsassessment of amino acid (or DNA) similarity and identity betweenmultiple proteins. Another non-limiting example of a mathematicalalgorithm utilized for the comparison of sequences is the algorithm ofMyers and Miller (1988) CABIOS 4:11-17. Such an algorithm isincorporated into the ALIGN program (version 2.0), which is part of theGCG Wisconsin Genetics Software Package, Version 10 (available fromAccelrys, Inc., 9685 Scranton Rd., San Diego, Calif., USA). Whenutilizing the ALIGN program for comparing amino acid sequences, a PAM120weight residue table, a gap length penalty of 12, and a gap penalty of 4can be used.

Unless otherwise stated, GAP Version 10, which uses the algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48(3):443-453, will be used todetermine sequence identity or similarity using the followingparameters: % identity and % similarity for a nucleotide sequence usingGAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoringmatrix; % identity or % similarity for an amino acid sequence using GAPweight of 8 and length weight of 2, and the BLOSUM62 scoring program.Equivalent programs may also be used. By “equivalent program” isintended any sequence comparison program that, for any two sequences inquestion, generates an alignment having identical nucleotide residuematches and an identical percent sequence identity when compared to thecorresponding alignment generated by GAP Version 10. The invention alsoencompasses variant nucleic acid molecules. “Variants” of the pesticidalprotein encoding nucleotide sequences include those sequences thatencode the pesticidal proteins disclosed herein but that differconservatively because of the degeneracy of the genetic code as well asthose that are sufficiently identical as discussed above. Naturallyoccurring allelic variants can be identified with the use of well-knownmolecular biology techniques, such as polymerase chain reaction (PCR)and hybridization techniques as outlined below. Variant nucleotidesequences also include synthetically derived nucleotide sequences thathave been generated, for example, by using site-directed mutagenesis butwhich still encode the pesticidal proteins disclosed in the presentinvention as discussed below. Variant proteins encompassed by thepresent invention are biologically active, that is they continue topossess the desired biological activity of the native protein, that is,retaining pesticidal activity. By “retains activity” is intended thatthe variant will have at least about 30%, at least about 50%, at leastabout 70%, or at least about 80% of the pesticidal activity of thenative protein. Methods for measuring pesticidal activity are well knownin the art. See, for example, Czapla and Lang (1990) J. Econ. Entomol.83: 2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206; Marrone etal. (1985) J. of Economic Entomology 78:290-293; and U.S. Pat. No.5,743,477, all of which are herein incorporated by reference in theirentirety.

The skilled artisan will further appreciate that changes can beintroduced by mutation of the nucleotide sequences of the inventionthereby leading to changes in the amino acid sequence of the encodedpesticidal proteins, without altering the biological activity of theproteins. Thus, variant isolated nucleic acid molecules can be createdby introducing one or more nucleotide substitutions, additions, ordeletions into the corresponding nucleotide sequence disclosed herein,such that one or more amino acid substitutions, additions or deletionsare introduced into the encoded protein. Mutations can be introduced bystandard techniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Such variant nucleotide sequences are also encompassed bythe present invention.

For example, conservative amino acid substitutions may be made at one ormore, predicted, nonessential amino acid residues. A “nonessential”amino acid residue is a residue that can be altered from the wild-typesequence of a pesticidal protein without altering the biologicalactivity, whereas an “essential” amino acid residue is required forbiological activity. A “conservative amino acid substitution” is one inwhich the amino acid residue is replaced with an amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art. These families includeamino acids with basic side chains (e.g., lysine, arginine, histidine),acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

Amino acid substitutions may be made in nonconserved regions that retainfunction. In general, such substitutions would not be made for conservedamino acid residues, or for amino acid residues residing within aconserved motif, where such residues are essential for protein activity.Examples of residues that are conserved and that may be essential forprotein activity include, for example, residues that are identicalbetween all proteins contained in an alignment of similar or relatedtoxins to the sequences of the invention (e.g., residues that areidentical in an alignment of homologous proteins). Examples of residuesthat are conserved but that may allow conservative amino acidsubstitutions and still retain activity include, for example, residuesthat have only conservative substitutions between all proteins containedin an alignment of similar or related toxins to the sequences of theinvention (e.g., residues that have only conservative substitutionsbetween all proteins contained in the alignment homologous proteins).However, one of skill in the art would understand that functionalvariants may have minor conserved or nonconserved alterations in theconserved residues.

Alternatively, variant nucleotide sequences can be made by introducingmutations randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forability to confer pesticidal activity to identify mutants that retainactivity. Following mutagenesis, the encoded protein can be expressedrecombinantly, and the activity of the protein can be determined usingstandard assay techniques.

Using methods such as PCR, hybridization, and the like correspondingpesticidal sequences can be identified, such sequences havingsubstantial identity to the sequences of the invention. See, forexample, Sambrook and Russell (2001) Molecular Cloning: A LaboratoryManual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)and Innis, et al. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, NY).

In a hybridization method, all or part of the pesticidal nucleotidesequence can be used to screen cDNA or genomic libraries. Methods forconstruction of such cDNA and genomic libraries are generally known inthe art and are disclosed in Sambrook and Russell, 2001, supra. Theso-called hybridization probes may be genomic DNA fragments, cDNAfragments, RNA fragments, or other oligonucleotides, and may be labeledwith a detectable group such as ³²P, or any other detectable marker,such as other radioisotopes, a fluorescent compound, an enzyme, or anenzyme co-factor. Probes for hybridization can be made by labelingsynthetic oligonucleotides based on the known pesticidalprotein-encoding nucleotide sequence disclosed herein. Degenerateprimers designed on the basis of conserved nucleotides or amino acidresidues in the nucleotide sequence or encoded amino acid sequence canadditionally be used. The probe typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12, at least about 25, at least about 50, 75, 100, 125, 150,175, or 200 consecutive nucleotides of nucleotide sequence encoding apesticidal protein of the invention or a fragment or variant thereof.Methods for the preparation of probes for hybridization are generallyknown in the art and are disclosed in Sambrook and Russell, 2001, supraherein incorporated by reference.

For example, an entire pesticidal protein sequence disclosed herein, orone or more portions thereof, may be used as a probe capable ofspecifically hybridizing to corresponding pesticidal protein-likesequences and messenger RNAs. To achieve specific hybridization under avariety of conditions, such probes include sequences that are unique andare preferably at least about 10 nucleotides in length, or at leastabout 20 nucleotides in length. Such probes may be used to amplifycorresponding pesticidal sequences from a chosen organism by PCR. Thistechnique may be used to isolate additional coding sequences from adesired organism or as a diagnostic assay to determine the presence ofcoding sequences in an organism. Hybridization techniques includehybridization screening of plated DNA libraries (either plaques orcolonies; see, for example, Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions. By “stringent conditions” or “stringent hybridizationconditions” is intended conditions under which a probe will hybridize toits target sequence to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,preferably less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4 to about 12 hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with ≧90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocolsin Molecular Biology, Chapter 2 (Greene Publishing andWiley-Interscience, New York). See Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.).

Isolated Proteins and Variants and Fragments Thereof

Pesticidal proteins are also encompassed within the present invention.By “pesticidal protein” is intended a protein having the amino acidsequence set forth in SEQ ID NO:2, 3, or 4. Fragments, biologicallyactive portions, and variants thereof (e.g., SEQ ID NO:5, 6, 7, and 8)are also provided, and may be used to practice the methods of thepresent invention. An “isolated protein” is used to refer to a proteinthat is no longer in its natural environment, for example in vitro or ina recombinant bacterial or plant host cell.

“Fragments” or “biologically active portions” include polypeptidefragments comprising amino acid sequences sufficiently identical to theamino acid sequence set forth in SEQ ID NO:2, 3, or 4, and that exhibitpesticidal activity. A biologically active portion of a pesticidalprotein can be a polypeptide that is, for example, 10, 25, 50, 100, 150,200, 250 or more amino acids in length. Such biologically activeportions can be prepared by recombinant techniques and evaluated forpesticidal activity. Methods for measuring pesticidal activity are wellknown in the art. See, for example, Czapla and Lang (1990) J. Econ.Entomol. 83:2480-2485; Andrews et al. (1988) Biochem. J. 252:199-206;Marrone et al. (1985) J. of Economic Entomology 78:290-293; and U.S.Pat. No. 5,743,477, all of which are herein incorporated by reference intheir entirety. As used here, a fragment comprises at least 8 contiguousamino acids of SEQ ID NO:2, 3, or 4. The invention encompasses otherfragments, however, such as any fragment in the protein greater thanabout 10, 20, 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550or more amino acids.

In some embodiments, the fragment is an N-terminal or a C-terminaltruncation of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25 or more amino acids relative to SEQ IDNO:2, 3, or 4 (e.g., SEQ ID NO:7 or 8). In some embodiments, thefragments encompassed herein result from the removal of the C-terminal1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25 or more amino acids, e.g., by proteolysis or by insertion of a stopcodon in the coding sequence.

By “variants” is intended proteins or polypeptides having an amino acidsequence that is at least about 60%, 65%, about 70%, 75%, about 80%,85%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identicalto the amino acid sequence of SEQ ID NO:2, 3, 4, 5, 6, 7, or 8. Variantsalso include polypeptides encoded by a nucleic acid molecule thathybridizes to the nucleic acid molecule of SEQ ID NO:1, 9, 10, or 11, ora complement thereof, under stringent conditions. Variants includepolypeptides that differ in amino acid sequence due to mutagenesis.Variant proteins encompassed by the present invention are biologicallyactive, that is they continue to possess the desired biological activityof the native protein, that is, retaining pesticidal activity. In someembodiments, the variants have improved activity. Methods for measuringpesticidal activity are well known in the art. See, for example, Czaplaand Lang (1990) J. Econ. Entomol. 83:2480-2485; Andrews et al. (1988)Biochem. J. 252:199-206; Marrone et al. (1985) J. of Economic Entomology78:290-293; and U.S. Pat. No. 5,743,477, all of which are hereinincorporated by reference in their entirety.

In some embodiments, the variant protein or polypeptide comprises one ormore substitutions at the amino acid positions selected from the groupconsisting of positions 307, 315, 317, 349, 351, 353, 355, 395, 399,407, 419, 435, 443, 465, 467, 483, 487, 495, 497, 499, 509, and 513relative to SEQ ID NO:2. In specific embodiments, the substitution is analanine for the native amino acid at the recited position(s). Alsoencompassed are the nucleotide sequence(s) encoding the variant proteinor polypeptide.

Bacterial genes, such as the axmi genes of this invention, quite oftenpossess multiple methionine initiation codons in proximity to the startof the open reading frame. Often, translation initiation at one or moreof these start codons will lead to generation of a functional protein.These start codons can include ATG codons. For example, SEQ ID NO:3 and4 represent alternate start site proteins encoded by SEQ ID NO:1.However, bacteria such as Bacillus sp. also recognize the codon GTG as astart codon, and proteins that initiate translation at GTG codonscontain a methionine at the first amino acid. On rare occasions,translation in bacterial systems can initiate at a TTG codon, though inthis event the TTG encodes a methionine. Furthermore, it is not oftendetermined a priori which of these codons are used naturally in thebacterium. Thus, it is understood that use of one of the alternatemethionine codons may also lead to generation of pesticidal proteins.These pesticidal proteins are encompassed in the present invention andmay be used in the methods of the present invention. It will beunderstood that, when expressed in plants, it will be necessary to alterthe alternate start codon to ATG for proper translation.

Antibodies to the polypeptides of the present invention, or to variantsor fragments thereof, are also encompassed. Methods for producingantibodies are well known in the art (see, for example, Harlow and Lane(1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.; U.S. Pat. No. 4,196,265).

Altered or Improved Variants

It is recognized that DNA sequences of a pesticidal protein may bealtered by various methods, and that these alterations may result in DNAsequences encoding proteins with amino acid sequences different thanthat encoded by a pesticidal protein of the present invention. Thisprotein may be altered in various ways including amino acidsubstitutions, deletions, truncations, and insertions of one or moreamino acids of SEQ ID NO:2, 3, or 4, including up to about 2, about 3,about 4, about 5, about 6, about 7, about 8, about 9, about 10, about15, about 20, about 25, about 30, about 35, about 40, about 45, about50, about 55, about 60, about 65, about 70, about 75, about 80, about85, about 90, about 100, about 105, about 110, about 115, about 120,about 125, about 130, about 135, about 140, about 145, about 150, about155, or more amino acid substitutions, deletions or insertions. Methodsfor such manipulations are generally known in the art. For example,amino acid sequence variants of a pesticidal protein can be prepared bymutations in the DNA. This may also be accomplished by one of severalforms of mutagenesis and/or in directed evolution. In some aspects, thechanges encoded in the amino acid sequence will not substantially affectthe function of the protein. Such variants will possess the desiredpesticidal activity. However, it is understood that the ability of apesticidal protein to confer pesticidal activity may be improved by theuse of such techniques upon the compositions of this invention. Forexample, one may express a pesticidal protein in host cells that exhibithigh rates of base misincorporation during DNA replication, such as XL-1Red (Stratagene, La Jolla, Calif.). After propagation in such strains,one can isolate the DNA (for example by preparing plasmid DNA, or byamplifying by PCR and cloning the resulting PCR fragment into a vector),culture the pesticidal protein mutations in a non-mutagenic strain, andidentify mutated genes with pesticidal activity, for example byperforming an assay to test for pesticidal activity. Generally, theprotein is mixed and used in feeding assays. See, for example Marrone etal. (1985) J. of Economic Entomology 78:290-293. Such assays can includecontacting plants with one or more pests and determining the plant'sability to survive and/or cause the death of the pests. Examples ofmutations that result in increased toxicity are found in Schnepf et al.(1998) Microbiol. Mol. Biol. Rev. 62:775-806.

Alternatively, alterations may be made to the protein sequence of manyproteins at the amino or carboxy terminus without substantiallyaffecting activity. This can include insertions, deletions, oralterations introduced by modern molecular methods, such as PCR,including PCR amplifications that alter or extend the protein codingsequence by virtue of inclusion of amino acid encoding sequences in theoligonucleotides utilized in the PCR amplification. Alternatively, theprotein sequences added can include entire protein-coding sequences,such as those used commonly in the art to generate protein fusions. Suchfusion proteins are often used to (1) increase expression of a proteinof interest (2) introduce a binding domain, enzymatic activity, orepitope to facilitate either protein purification, protein detection, orother experimental uses known in the art (3) target secretion ortranslation of a protein to a subcellular organelle, such as theperiplasmic space of Gram-negative bacteria, or the endoplasmicreticulum of eukaryotic cells, the latter of which often results inglycosylation of the protein.

Variant nucleotide and amino acid sequences of the present inventionalso encompass sequences derived from mutagenic and recombinogenicprocedures such as DNA shuffling. With such a procedure, one or moredifferent pesticidal protein coding regions can be used to create a newpesticidal protein possessing the desired properties. In this manner,libraries of recombinant polynucleotides are generated from a populationof related sequence polynucleotides comprising sequence regions thathave substantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between a pesticidal geneof the invention and other known pesticidal genes to obtain a new genecoding for a protein with an improved property of interest, such as anincreased insecticidal activity. Strategies for such DNA shuffling areknown in the art. See, for example, Stemmer (1994) Proc. Natl. Acad.Sci. USA 91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri etal. (1997) Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol.272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

Domain swapping or shuffling is another mechanism for generating alteredpesticidal proteins. Domains may be swapped between pesticidal proteins,resulting in hybrid or chimeric toxins with improved pesticidal activityor target spectrum. Methods for generating recombinant proteins andtesting them for pesticidal activity are well known in the art (see, forexample, Naimov et al. (2001) Appl. Environ. Microbiol. 67:5328-5330; deMaagd et al. (1996) Appl. Environ. Microbiol. 62:1537-1543; Ge et al.(1991) J. Biol. Chem. 266:17954-17958; Schnepf et al. (1990) J. Biol.Chem. 265:20923-20930; Rang et al. 91999) Appl. Environ. Microbiol.65:2918-2925).

Vectors

A pesticidal sequence of the invention may be provided in an expressioncassette for expression in a plant of interest. By “plant expressioncassette” is intended a DNA construct that is capable of resulting inthe expression of a protein from an open reading frame in a plant cell.Typically these contain a promoter and a coding sequence. Often, suchconstructs will also contain a 3′ untranslated region. Such constructsmay contain a “signal sequence” or “leader sequence” to facilitateco-translational or post-translational transport of the peptide tocertain intracellular structures such as the chloroplast (or otherplastid), endoplasmic reticulum, or Golgi apparatus.

By “signal sequence” is intended a sequence that is known or suspectedto result in cotranslational or post-translational peptide transportacross the cell membrane. In eukaryotes, this typically involvessecretion into the Golgi apparatus, with some resulting glycosylation.Insecticidal toxins of bacteria are often synthesized as protoxins,which are protolytically activated in the gut of the target pest (Chang(1987) Methods Enzymol. 153:507-516). In some embodiments of the presentinvention, the signal sequence is located in the native sequence, or maybe derived from a sequence of the invention. By “leader sequence” isintended any sequence that when translated, results in an amino acidsequence sufficient to trigger co-translational transport of the peptidechain to a subcellular organelle. Thus, this includes leader sequencestargeting transport and/or glycosylation by passage into the endoplasmicreticulum, passage to vacuoles, plastids including chloroplasts,mitochondria, and the like.

By “plant transformation vector” is intended a DNA molecule that isnecessary for efficient transformation of a plant cell. Such a moleculemay consist of one or more plant expression cassettes, and may beorganized into more than one “vector” DNA molecule. For example, binaryvectors are plant transformation vectors that utilize two non-contiguousDNA vectors to encode all requisite cis- and trans-acting functions fortransformation of plant cells (Hellens and Mullineaux (2000) Trends inPlant Science 5:446-451). “Vector” refers to a nucleic acid constructdesigned for transfer between different host cells. “Expression vector”refers to a vector that has the ability to incorporate, integrate andexpress heterologous DNA sequences or fragments in a foreign cell. Thecassette will include 5′ and 3′ regulatory sequences operably linked toa sequence of the invention. By “operably linked” is intended afunctional linkage between a promoter and a second sequence, wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand, where necessary to join two protein coding regions, contiguous andin the same reading frame. The cassette may additionally contain atleast one additional gene to be cotransformed into the organism.Alternatively, the additional gene(s) can be provided on multipleexpression cassettes.

“Promoter” refers to a nucleic acid sequence that functions to directtranscription of a downstream coding sequence. The promoter togetherwith other transcriptional and translational regulatory nucleic acidsequences (also termed “control sequences”) are necessary for theexpression of a DNA sequence of interest.

Such an expression cassette is provided with a plurality of restrictionsites for insertion of the pesticidal sequence to be under thetranscriptional regulation of the regulatory regions.

The expression cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a DNA sequence of the invention, and a translationaland transcriptional termination region (i.e., termination region)functional in plants. The promoter may be native or analogous, orforeign or heterologous, to the plant host and/or to the DNA sequence ofthe invention. Additionally, the promoter may be the natural sequence oralternatively a synthetic sequence. Where the promoter is “native” or“homologous” to the plant host, it is intended that the promoter isfound in the native plant into which the promoter is introduced. Wherethe promoter is “foreign” or “heterologous” to the DNA sequence of theinvention, it is intended that the promoter is not the native ornaturally occurring promoter for the operably linked DNA sequence of theinvention.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,may be native with the plant host, or may be derived from another source(i.e., foreign or heterologous to the promoter, the DNA sequence ofinterest, the plant host, or any combination thereof). Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase termination regions.See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot(1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149;Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; andJoshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

Where appropriate, the gene(s) may be optimized for increased expressionin the transformed host cell. That is, the genes can be synthesizedusing host cell-preferred codons for improved expression, or may besynthesized using codons at a host-preferred codon usage frequency.Generally, the GC content of the gene will be increased. See, forexample, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for adiscussion of host-preferred codon usage. Methods are available in theart for synthesizing plant-preferred genes. See, for example, U.S. Pat.Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic AcidsRes. 17:477-498, herein incorporated by reference.

In one embodiment, the pesticidal protein is targeted to the chloroplastfor expression. In this manner, where the pesticidal protein is notdirectly inserted into the chloroplast, the expression cassette willadditionally contain a nucleic acid encoding a transit peptide to directthe pesticidal protein to the chloroplasts. Such transit peptides areknown in the art. See, for example, Von Heijne et al. (1991) Plant Mol.Biol. Rep. 9:104-126; Clark et al. (1989) J. Biol. Chem.264:17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84:965-968;Romer et al. (1993) Biochem. Biophys. Res. Commun. 196:1414-1421; andShah et al. (1986) Science 233:478-481.

The pesticidal gene to be targeted to the chloroplast may be optimizedfor expression in the chloroplast to account for differences in codonusage between the plant nucleus and this organelle. In this manner, thenucleic acids of interest may be synthesized using chloroplast-preferredcodons. See, for example, U.S. Pat. No. 5,380,831, herein incorporatedby reference.

Plant Transformation

Methods of the invention involve introducing a nucleotide construct intoa plant. By “introducing” is intended to present to the plant thenucleotide construct in such a manner that the construct gains access tothe interior of a cell of the plant. The methods of the invention do notrequire that a particular method for introducing a nucleotide constructto a plant is used, only that the nucleotide construct gains access tothe interior of at least one cell of the plant. Methods for introducingnucleotide constructs into plants are known in the art including, butnot limited to, stable transformation methods, transient transformationmethods, and virus-mediated methods.

By “plant” is intended whole plants, plant organs (e.g., leaves, stems,roots, etc.), seeds, plant cells, propagules, embryos and progeny of thesame. Plant cells can be differentiated or undifferentiated (e.g.callus, suspension culture cells, protoplasts, leaf cells, root cells,phloem cells, pollen).

“Transgenic plants” or “transformed plants” or “stably transformed”plants or cells or tissues refers to plants that have incorporated orintegrated exogenous nucleic acid sequences or DNA fragments into theplant cell. These nucleic acid sequences include those that areexogenous, or not present in the untransformed plant cell, as well asthose that may be endogenous, or present in the untransformed plantcell. “Heterologous” generally refers to the nucleic acid sequences thatare not endogenous to the cell or part of the native genome in whichthey are present, and have been added to the cell by infection,transfection, microinjection, electroporation, microprojection, or thelike.

The transgenic plants of the invention express one or more of thepesticidal sequences disclosed herein. In various embodiments, thetransgenic plant further comprises one or more additional genes forinsect resistance, for example, one or more additional genes forcontrolling coleopteran, lepidopteran, heteropteran, or nematode pests.It will be understood by one of skill in the art that the transgenicplant may comprise any gene imparting an agronomic trait of interest.

Transformation of plant cells can be accomplished by one of severaltechniques known in the art. The pesticidal gene of the invention may bemodified to obtain or enhance expression in plant cells. Typically aconstruct that expresses such a protein would contain a promoter todrive transcription of the gene, as well as a 3′ untranslated region toallow transcription termination and polyadenylation. The organization ofsuch constructs is well known in the art. In some instances, it may beuseful to engineer the gene such that the resulting peptide is secreted,or otherwise targeted within the plant cell. For example, the gene canbe engineered to contain a signal peptide to facilitate transfer of thepeptide to the endoplasmic reticulum. It may also be preferable toengineer the plant expression cassette to contain an intron, such thatmRNA processing of the intron is required for expression.

Typically this “plant expression cassette” will be inserted into a“plant transformation vector”. This plant transformation vector may becomprised of one or more DNA vectors needed for achieving planttransformation. For example, it is a common practice in the art toutilize plant transformation vectors that are comprised of more than onecontiguous DNA segment. These vectors are often referred to in the artas “binary vectors”. Binary vectors as well as vectors with helperplasmids are most often used for Agrobacterium-mediated transformation,where the size and complexity of DNA segments needed to achieveefficient transformation is quite large, and it is advantageous toseparate functions onto separate DNA molecules. Binary vectors typicallycontain a plasmid vector that contains the cis-acting sequences requiredfor T-DNA transfer (such as left border and right border), a selectablemarker that is engineered to be capable of expression in a plant cell,and a “gene of interest” (a gene engineered to be capable of expressionin a plant cell for which generation of transgenic plants is desired).Also present on this plasmid vector are sequences required for bacterialreplication. The cis-acting sequences are arranged in a fashion to allowefficient transfer into plant cells and expression therein. For example,the selectable marker gene and the pesticidal gene are located betweenthe left and right borders. Often a second plasmid vector contains thetrans-acting factors that mediate T-DNA transfer from Agrobacterium toplant cells. This plasmid often contains the virulence functions (Virgenes) that allow infection of plant cells by Agrobacterium, andtransfer of DNA by cleavage at border sequences and vir-mediated DNAtransfer, as is understood in the art (Hellens and Mullineaux (2000)Trends in Plant Science 5:446-451). Several types of Agrobacteriumstrains (e.g. LBA4404, GV3101, EHA101, EHA105, etc.) can be used forplant transformation. The second plasmid vector is not necessary fortransforming the plants by other methods such as microprojection,microinjection, electroporation, polyethylene glycol, etc.

In general, plant transformation methods involve transferringheterologous DNA into target plant cells (e.g. immature or matureembryos, suspension cultures, undifferentiated callus, protoplasts,etc.), followed by applying a maximum threshold level of appropriateselection (depending on the selectable marker gene) to recover thetransformed plant cells from a group of untransformed cell mass.Explants are typically transferred to a fresh supply of the same mediumand cultured routinely. Subsequently, the transformed cells aredifferentiated into shoots after placing on regeneration mediumsupplemented with a maximum threshold level of selecting agent. Theshoots are then transferred to a selective rooting medium for recoveringrooted shoot or plantlet. The transgenic plantlet then grows into amature plant and produces fertile seeds (e.g. Hiei et al. (1994) ThePlant Journal 6:271-282; Ishida et al. (1996) Nature Biotechnology14:745-750). Explants are typically transferred to a fresh supply of thesame medium and cultured routinely. A general description of thetechniques and methods for generating transgenic plants are found inAyres and Park (1994) Critical Reviews in Plant Science 13:219-239 andBommineni and Jauhar (1997) Maydica 42:107-120. Since the transformedmaterial contains many cells; both transformed and non-transformed cellsare present in any piece of subjected target callus or tissue or groupof cells. The ability to kill non-transformed cells and allowtransformed cells to proliferate results in transformed plant cultures.Often, the ability to remove non-transformed cells is a limitation torapid recovery of transformed plant cells and successful generation oftransgenic plants.

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Generation oftransgenic plants may be performed by one of several methods, including,but not limited to, microinjection, electroporation, direct genetransfer, introduction of heterologous DNA by Agrobacterium into plantcells (Agrobacterium-mediated transformation), bombardment of plantcells with heterologous foreign DNA adhered to particles, ballisticparticle acceleration, aerosol beam transformation (U.S. PublishedApplication No. 20010026941; U.S. Pat. No. 4,945,050; InternationalPublication No. WO 91/00915; U.S. Published Application No. 2002015066),Lec1 transformation, and various other non-particle direct-mediatedmethods to transfer DNA.

Methods for transformation of chloroplasts are known in the art. See,for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87:8526-8530;Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90:913-917; Svab andMaliga (1993) EMBO J. 12:601-606. The method relies on particle gundelivery of DNA containing a selectable marker and targeting of the DNAto the plastid genome through homologous recombination. Additionally,plastid transformation can be accomplished by transactivation of asilent plastid-borne transgene by tissue-preferred expression of anuclear-encoded and plastid-directed RNA polymerase. Such a system hasbeen reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA91:7301-7305.

Following integration of heterologous foreign DNA into plant cells, onethen applies a maximum threshold level of appropriate selection in themedium to kill the untransformed cells and separate and proliferate theputatively transformed cells that survive from this selection treatmentby transferring regularly to a fresh medium. By continuous passage andchallenge with appropriate selection, one identifies and proliferatesthe cells that are transformed with the plasmid vector. Molecular andbiochemical methods can then be used to confirm the presence of theintegrated heterologous gene of interest into the genome of thetransgenic plant.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a nucleotide construct of theinvention, for example, an expression cassette of the invention, stablyincorporated into their genome.

Evaluation of Plant Transformation

Following introduction of heterologous foreign DNA into plant cells, thetransformation or integration of heterologous gene in the plant genomeis confirmed by various methods such as analysis of nucleic acids,proteins and metabolites associated with the integrated gene.

PCR analysis is a rapid method to screen transformed cells, tissue orshoots for the presence of incorporated gene at the earlier stage beforetransplanting into the soil (Sambrook and Russell (2001) MolecularCloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). PCR is carried out using oligonucleotide primersspecific to the gene of interest or Agrobacterium vector background,etc.

Plant transformation may be confirmed by Southern blot analysis ofgenomic DNA (Sambrook and Russell, 2001, supra). In general, total DNAis extracted from the transformant, digested with appropriaterestriction enzymes, fractionated in an agarose gel and transferred to anitrocellulose or nylon membrane. The membrane or “blot” is then probedwith, for example, radiolabeled ³²P target DNA fragment to confirm theintegration of introduced gene into the plant genome according tostandard techniques (Sambrook and Russell, 2001, supra).

In Northern blot analysis, RNA is isolated from specific tissues oftransformant, fractionated in a formaldehyde agarose gel, and blottedonto a nylon filter according to standard procedures that are routinelyused in the art (Sambrook and Russell, 2001, supra). Expression of RNAencoded by the pesticidal gene is then tested by hybridizing the filterto a radioactive probe derived from a pesticidal gene, by methods knownin the art (Sambrook and Russell, 2001, supra).

Western blot, biochemical assays and the like may be carried out on thetransgenic plants to confirm the presence of protein encoded by thepesticidal gene by standard procedures (Sambrook and Russell, 2001,supra) using antibodies that bind to one or more epitopes present on thepesticidal protein.

Pesticidal Activity in Plants

In another aspect of the invention, one may generate transgenic plantsexpressing a pesticidal protein that has pesticidal activity. Methodsdescribed above by way of example may be utilized to generate transgenicplants, but the manner in which the transgenic plant cells are generatedis not critical to this invention. Methods known or described in the artsuch as Agrobacterium-mediated transformation, biolistic transformation,and non-particle-mediated methods may be used at the discretion of theexperimenter. Plants expressing a pesticidal protein may be isolated bycommon methods described in the art, for example by transformation ofcallus, selection of transformed callus, and regeneration of fertileplants from such transgenic callus. In such process, one may use anygene as a selectable marker so long as its expression in plant cellsconfers ability to identify or select for transformed cells.

A number of markers have been developed for use with plant cells, suchas resistance to chloramphenicol, the aminoglycoside G418, hygromycin,or the like. Other genes that encode a product involved in chloroplastmetabolism may also be used as selectable markers. For example, genesthat provide resistance to plant herbicides such as glyphosate,bromoxynil, or imidazolinone may find particular use. Such genes havebeen reported (Stalker et al. (1985) J. Biol. Chem. 263:6310-6314(bromoxynil resistance nitrilase gene); and Sathasivan et al. (1990)Nucl. Acids Res. 18:2188 (AHAS imidazolinone resistance gene).Additionally, the genes disclosed herein are useful as markers to assesstransformation of bacterial or plant cells. Methods for detecting thepresence of a transgene in a plant, plant organ (e.g., leaves, stems,roots, etc.), seed, plant cell, propagule, embryo or progeny of the sameare well known in the art. In one embodiment, the presence of thetransgene is detected by testing for pesticidal activity.

Fertile plants expressing a pesticidal protein may be tested forpesticidal activity, and the plants showing optimal activity selectedfor further breeding. Methods are available in the art to assay for pestactivity. Generally, the protein is mixed and used in feeding assays.See, for example Marrone et al. (1985) J. of Economic Entomology78:290-293.

The present invention may be used for transformation of any plantspecies, including, but not limited to, monocots and dicots. Examples ofplants of interest include, but are not limited to, corn (maize),sorghum, wheat, sunflower, tomato, crucifers, peppers, potato, cotton,rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape,Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato,cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana,avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond,oats, vegetables, ornamentals, and conifers.

Vegetables include, but are not limited to, tomatoes, lettuce, greenbeans, lima beans, peas, and members of the genus Curcumis such ascucumber, cantaloupe, and musk melon. Ornamentals include, but are notlimited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils,petunias, carnation, poinsettia, and chrysanthemum. Preferably, plantsof the present invention are crop plants (for example, maize, sorghum,wheat, sunflower, tomato, crucifers, peppers, potato, cotton, rice,soybean, sugarbeet, sugarcane, tobacco, barley, oilseed rape, etc.).

Use in Pesticidal Control

General methods for employing strains comprising a nucleotide sequenceof the present invention, or a variant thereof, in pesticide control orin engineering other organisms as pesticidal agents are known in theart. See, for example U.S. Pat. No. 5,039,523 and EP 0480762A2.

The Bacillus strains containing a nucleotide sequence of the presentinvention, or a variant thereof, or the microorganisms that have beengenetically altered to contain a pesticidal gene and protein may be usedfor protecting agricultural crops and products from pests. In one aspectof the invention, whole, i.e., unlysed, cells of a toxin(pesticide)-producing organism are treated with reagents that prolongthe activity of the toxin produced in the cell when the cell is appliedto the environment of target pest(s).

Alternatively, the pesticide is produced by introducing a pesticidalgene into a cellular host. Expression of the pesticidal gene results,directly or indirectly, in the intracellular production and maintenanceof the pesticide. In one aspect of this invention, these cells are thentreated under conditions that prolong the activity of the toxin producedin the cell when the cell is applied to the environment of targetpest(s). The resulting product retains the toxicity of the toxin. Thesenaturally encapsulated pesticides may then be formulated in accordancewith conventional techniques for application to the environment hostinga target pest, e.g., soil, water, and foliage of plants. See, forexample EPA 0192319, and the references cited therein. Alternatively,one may formulate the cells expressing a gene of this invention such asto allow application of the resulting material as a pesticide.

Pesticidal Compositions

The active ingredients of the present invention are normally applied inthe form of compositions and can be applied to the crop area or plant tobe treated, simultaneously or in succession, with other compounds. Thesecompounds can be fertilizers, weed killers, cryoprotectants,surfactants, detergents, pesticidal soaps, dormant oils, polymers,and/or time-release or biodegradable carrier formulations that permitlong-term dosing of a target area following a single application of theformulation. They can also be selective herbicides, chemicalinsecticides, virucides, microbicides, amoebicides, pesticides,fungicides, bacteriocides, nematocides, molluscicides or mixtures ofseveral of these preparations, if desired, together with furtheragriculturally acceptable carriers, surfactants or application-promotingadjuvants customarily employed in the art of formulation. Suitablecarriers and adjuvants can be solid or liquid and correspond to thesubstances ordinarily employed in formulation technology, e.g. naturalor regenerated mineral substances, solvents, dispersants, wettingagents, tackifiers, binders or fertilizers Likewise the formulations maybe prepared into edible “baits” or fashioned into pest “traps” to permitfeeding or ingestion by a target pest of the pesticidal formulation.

Methods of applying an active ingredient of the present invention or anagrochemical composition of the present invention that contains at leastone of the pesticidal proteins produced by the bacterial strains of thepresent invention include leaf application, seed coating and soilapplication. The number of applications and the rate of applicationdepend on the intensity of infestation by the corresponding pest.

The composition may be formulated as a powder, dust, pellet, granule,spray, emulsion, colloid, solution, or such like, and may be prepared bysuch conventional means as desiccation, lyophilization, homogenation,extraction, filtration, centrifugation, sedimentation, or concentrationof a culture of cells comprising the polypeptide. In all suchcompositions that contain at least one such pesticidal polypeptide, thepolypeptide may be present in a concentration of from about 1% to about99% by weight.

Lepidopteran, dipteran, heteropteran, nematode, or coleopteran pests maybe killed or reduced in numbers in a given area by the methods of theinvention, or may be prophylactically applied to an environmental areato prevent infestation by a susceptible pest. Preferably the pestingests, or is contacted with, a pesticidally-effective amount of thepolypeptide. By “pesticidally-effective amount” is intended an amount ofthe pesticide that is able to bring about death to at least one pest, orto noticeably reduce pest growth, feeding, or normal physiologicaldevelopment. This amount will vary depending on such factors as, forexample, the specific target pests to be controlled, the specificenvironment, location, plant, crop, or agricultural site to be treated,the environmental conditions, and the method, rate, concentration,stability, and quantity of application of the pesticidally-effectivepolypeptide composition. The formulations may also vary with respect toclimatic conditions, environmental considerations, and/or frequency ofapplication and/or severity of pest infestation.

The pesticide compositions described may be made by formulating eitherthe bacterial cell, crystal and/or spore suspension, or isolated proteincomponent with the desired agriculturally-acceptable carrier. Thecompositions may be formulated prior to administration in an appropriatemeans such as lyophilized, freeze-dried, desiccated, or in an aqueouscarrier, medium or suitable diluent, such as saline or other buffer. Theformulated compositions may be in the form of a dust or granularmaterial, or a suspension in oil (vegetable or mineral), or water oroil/water emulsions, or as a wettable powder, or in combination with anyother carrier material suitable for agricultural application. Suitableagricultural carriers can be solid or liquid and are well known in theart. The term “agriculturally-acceptable carrier” covers all adjuvants,inert components, dispersants, surfactants, tackifiers, binders, etc.that are ordinarily used in pesticide formulation technology; these arewell known to those skilled in pesticide formulation. The formulationsmay be mixed with one or more solid or liquid adjuvants and prepared byvarious means, e.g., by homogeneously mixing, blending and/or grindingthe pesticidal composition with suitable adjuvants using conventionalformulation techniques. Suitable formulations and application methodsare described in U.S. Pat. No. 6,468,523, herein incorporated byreference.

The plants can also be treated with one or more chemical compositions,including one or more herbicide, insecticides, or fungicides. Exemplarychemical compositions include: Fruits/Vegetables Herbicides: Atrazine,Bromacil, Diuron, Glyphosate, Linuron, Metribuzin, Simazine,Trifluralin, Fluazifop, Glufosinate, Halo sulfuron Gowan, Paraquat,Propyzamide, Sethoxydim, Butafenacil, Halosulfuron, Indaziflam;Fruits/Vegetables Insecticides: Aldicarb, Bacillus thuriengiensis,Carbaryl, Carbofuran, Chlorpyrifos, Cypermethrin, Deltamethrin,Diazinon, Malathion, Abamectin, Cyfluthrin/beta-cyfluthrin,Esfenvalerate, Lambda-cyhalothrin, Acequinocyl, Bifenazate,Methoxyfenozide, Novaluron, Chromafenozide, Thiacloprid, Dinotefuran,Fluacrypyrim, Tolfenpyrad, Clothianidin, Spirodiclofen,Gamma-cyhalothrin, Spiromesifen, Spinosad, Rynaxypyr, Cyazypyr,Spinoteram, Triflumuron, Spirotetramat, Imidacloprid, Flubendiamide,Thiodicarb, Metaflumizone, Sulfoxaflor, Cyflumetofen, Cyanopyrafen,Imidacloprid, Clothianidin, Thiamethoxam, Spinotoram, Thiodicarb,Flonicamid, Methiocarb, Emamectin-benzoate, Indoxacarb, Forthiazate,Fenamiphos, Cadusaphos, Pyriproxifen, Fenbutatin-oxid, Hexthiazox,Methomyl,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on;Fruits/Vegetables Fungicides: Carbendazim, Chlorothalonil, EBDCs,Sulphur, Thiophanate-methyl, Azoxystrobin, Cymoxanil, Fluazinam,Fosetyl, Iprodione, Kresoxim-methyl, Metalaxyl/mefenoxam,Trifloxystrobin, Ethaboxam, Iprovalicarb, Trifloxystrobin, Fenhexamid,Oxpoconazole fumarate, Cyazofamid, Fenamidone, Zoxamide, Picoxystrobin,Pyraclostrobin, Cyflufenamid, Boscalid; Cereals Herbicides: Isoproturon,Bromoxynil, Ioxynil, Phenoxies, Chlorsulfuron, Clodinafop, Diclofop,Diflufenican, Fenoxaprop, Florasulam, Fluoroxypyr, Metsulfuron,Triasulfuron, Flucarbazone, Iodosulfuron, Propoxycarbazone, Picolinafen,Mesosulfuron, Beflubutamid, Pinoxaden, Amidosulfuron, Thifensulfuron,Tribenuron, Flupyrsulfuron, Sulfosulfuron, Pyrasulfotole, Pyroxsulam,Flufenacet, Tralkoxydim, Pyroxasulfon; Cereals Fungicides: Carbendazim,Chlorothalonil, Azoxystrobin, Cyproconazole, Cyprodinil, Fenpropimorph,Epoxiconazole, Kresoxim-methyl, Quinoxyfen, Tebuconazole,Trifloxystrobin, Simeconazole, Picoxystrobin, Pyraclostrobin,Dimoxystrobin, Prothioconazole, Fluoxastrobin; Cereals Insecticides:Dimethoate, Lambda-cyhalthrin, Deltamethrin, alpha-Cypermethrin,β-cyfluthrin, Bifenthrin, Imidacloprid, Clothianidin, Thiamethoxam,Thiacloprid, Acetamiprid, Dinetofuran, Clorphyriphos, Metamidophos,Oxidemethon-methyl, Pirimicarb, Methiocarb; Maize Herbicides: Atrazine,Alachlor, Bromoxynil, Acetochlor, Dicamba, Clopyralid, (S-)Dimethenamid,Glufosinate, Glyphosate, Isoxaflutole, (S-)Metolachlor, Mesotrione,Nicosulfuron, Primisulfuron, Rimsulfuron, Sulcotrione, Foramsulfuron,Topramezone, Tembotrione, Saflufenacil, Thiencarbazone, Flufenacet,Pyroxasulfon; Maize Insecticides: Carbofuran, Chlorpyrifos, Bifenthrin,Fipronil, Imidacloprid, Lambda-Cyhalothrin, Tefluthrin, Terbufos,Thiamethoxam, Clothianidin, Spiromesifen, Flubendiamide, Triflumuron,Rynaxypyr, Deltamethrin, Thiodicarb, β-Cyfluthrin, Cypermethrin,Bifenthrin, Lufenuron, Triflumoron, Tefluthrin, Tebupirimphos,Ethiprole, Cyazypyr, Thiacloprid, Acetamiprid, Dinetofuran, Avermectin,Methiocarb, Spirodiclofen, Spirotetramat; Maize Fungicides: Fenitropan,Thiram, Prothioconazole, Tebuconazole, Trifloxystrobin; Rice Herbicides:Butachlor, Propanil, Azimsulfuron, Bensulfuron, Cyhalofop, Daimuron,Fentrazamide, Imazosulfuron, Mefenacet, Oxaziclomefone, Pyrazosulfuron,Pyributicarb, Quinclorac, Thiobencarb, Indanofan, Flufenacet,Fentrazamide, Halosulfuron, Oxaziclomefone, Benzobicyclon, Pyriftalid,Penoxsulam, Bispyribac, Oxadiargyl, Ethoxysulfuron, Pretilachlor,Mesotrione, Tefuryltrione, Oxadiazone, Fenoxaprop, Pyrimisulfan; RiceInsecticides: Diazinon, Fenitrothion, Fenobucarb, Monocrotophos,Benfuracarb, Buprofezin, Dinotefuran, Fipronil, Imidacloprid,Isoprocarb, Thiacloprid, Chromafenozide, Thiacloprid, Dinotefuran,Clothianidin, Ethiprole, Flubendiamide, Rynaxypyr, Deltamethrin,Acetamiprid, Thiamethoxam, Cyazypyr, Spinosad, Spinotoram,Emamectin-Benzoate, Cypermethrin, Chlorpyriphos, Cartap, Methamidophos,Etofenprox, Triazophos,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Carbofuran, Benfuracarb; Rice Fungicides: Thiophanate-methyl,Azoxystrobin, Carpropamid, Edifenphos, Ferimzone, Iprobenfos,Isoprothiolane, Pencycuron, Probenazole, Pyroquilon, Tricyclazole,Trifloxystrobin, Diclocymet, Fenoxanil, Simeconazole, Tiadinil; CottonHerbicides: Diuron, Fluometuron, MSMA, Oxyfluorfen, Prometryn,Trifluralin, Carfentrazone, Clethodim, Fluazifop-butyl, Glyphosate,Norflurazon, Pendimethalin, Pyrithiobac-sodium, Trifloxysulfuron,Tepraloxydim, Glufosinate, Flumioxazin, Thidiazuron; CottonInsecticides: Acephate, Aldicarb, Chlorpyrifos, Cypermethrin,Deltamethrin, Malathion, Monocrotophos, Abamectin, Acetamiprid,Emamectin Benzoate, Imidacloprid, Indoxacarb, Lambda-Cyhalothrin,Spinosad, Thiodicarb, Gamma-Cyhalothrin, Spiromesifen, Pyridalyl,Flonicamid, Flubendiamide, Triflumuron, Rynaxypyr, Beta-Cyfluthrin,Spirotetramat, Clothianidin, Thiamethoxam, Thiacloprid, Dinetofuran,Flubendiamide, Cyazypyr, Spinosad, Spinotoram, gamma Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Thiodicarb, Avermectin, Flonicamid, Pyridalyl, Spiromesifen,Sulfoxaflor, Profenophos, Thriazophos, Endosulfan; Cotton Fungicides:Etridiazole, Metalaxyl, Quintozene; Soybean Herbicides: Alachlor,Bentazone, Trifluralin, Chlorimuron-Ethyl, Cloransulam-Methyl,Fenoxaprop, Fomesafen, Fluazifop, Glyphosate, Imazamox, Imazaquin,Imazethapyr, (S-)Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim,Glufosinate; Soybean Insecticides: Lambda-cyhalothrin, Methomyl,Parathion, Thiocarb, Imidacloprid, Clothianidin, Thiamethoxam,Thiacloprid, Acetamiprid, Dinetofuran, Flubendiamide, Rynaxypyr,Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Fipronil, Ethiprole,Deltamethrin, β-Cyfluthrin, gamma and lambda Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Spirotetramat, Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb,beta-Cyfluthrin; Soybean Fungicides: Azoxystrobin, Cyproconazole,Epoxiconazole, Flutriafol, Pyraclostrobin, Tebuconazole,Trifloxystrobin, Prothioconazole, Tetraconazole; Sugarbeet Herbicides:Chloridazon, Desmedipham, Ethofumesate, Phenmedipham, Triallate,Clopyralid, Fluazifop, Lenacil, Metamitron, Quinmerac, Cycloxydim,Triflusulfuron, Tepraloxydim, Quizalofop; Sugarbeet Insecticides:Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid,Dinetofuran, Deltamethrin, β-Cyfluthrin, gamma/lambda Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Tefluthrin, Rynaxypyr, Cyaxypyr, Fipronil, Carbofuran; CanolaHerbicides: Clopyralid, Diclofop, Fluazifop, Glufosinate, Glyphosate,Metazachlor, Trifluralin Ethametsulfuron, Quinmerac, Quizalofop,Clethodim, Tepraloxydim; Canola Fungicides: Azoxystrobin, Carbendazim,Fludioxonil, Iprodione, Prochloraz, Vinclozolin; Canola Insecticides:

Carbofuran, Organophosphates, Pyrethroids, Thiacloprid, Deltamethrin,Imidacloprid, Clothianidin, Thiamethoxam, Acetamiprid, Dinetofuran,β-Cyfluthrin, gamma and lambda Cyhalothrin, tau-Fluvaleriate, Ethiprole,Spinosad, Spinotoram, Flubendiamide, Rynaxypyr, Cyazypyr,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on.

“Pest” includes but is not limited to, insects, fungi, bacteria,nematodes, mites, ticks, and the like. Insect pests include insectsselected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera,Mallophaga, Homoptera, Hemiptera, Orthroptera, Thysanoptera, Dermaptera,Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularlyColeoptera, Lepidoptera, and Diptera.

The order Coleoptera includes the suborders Adephaga and Polyphaga.Suborder Adephaga includes the superfamilies Caraboidea and Gyrinoidea,while suborder Polyphaga includes the superfamilies Hydrophiloidea,Staphylinoidea, Cantharoidea, Cleroidea, Elateroidea, Dascilloidea,Dryopoidea, Byrrhoidea, Cucujoidea, Meloidea, Mordelloidea,Tenebrionoidea, Bostrichoidea, Scarabaeoidea, Cerambycoidea,Chrysomeloidea, and Curculionoidea. Superfamily Caraboidea includes thefamilies Cicindelidae, Carabidae, and Dytiscidae. Superfamily Gyrinoideaincludes the family Gyrimidae. Superfamily Hydrophiloidea includes thefamily Hydrophilidae. Superfamily Staphylinoidea includes the familiesSilphidae and Staphylimidae. Superfamily Cantharoidea includes thefamilies Cantharidae and Lampyridae. Superfamily Cleroidea includes thefamilies Cleridae and Dermestidae. Superfamily Elateroidea includes thefamilies Elateridae and Buprestidae. Superfamily Cucujoidea includes thefamily Coccinellidae. Superfamily Meloidea includes the family Meloidae.Superfamily Tenebrionoidea includes the family Tenebrionidae.Superfamily Scarabaeoidea includes the families Passalidae andScarabaeidae. Superfamily Cerambycoidea includes the familyCerambycidae. Superfamily Chrysomeloidea includes the familyChrysomelidae. Superfamily Curculionoidea includes the familiesCurculionidae and Scolytidae.

The order Diptera includes the Suborders Nematocera, Brachycera, andCyclorrhapha. Suborder Nematocera includes the families Tipulidae,Psychodidae, Culicidae, Ceratopogonidae, Chironomidae, Simuliidae,Bibionidae, and Cecidomyiidae. Suborder Brachycera includes the familiesStratiomyidae, Tabanidae, Therevidae, Asilidae, Mydidae, Bombyliidae,and Dolichopodidae. Suborder Cyclorrhapha includes the Divisions Aschizaand Aschiza. Division Aschiza includes the families Phoridae, Syrphidae,and Conopidae. Division Aschiza includes the Sections Acalyptratae andCalyptratae. Section Acalyptratae includes the families Otitidae,Tephritidae, Agromyzidae, and Drosophilidae. Section Calyptrataeincludes the families Hippoboscidae, Oestridae, Tachinidae,Anthomyiidae, Muscidae, Calliphoridae, and Sarcophagidae.

The order Lepidoptera includes the families Papilionidae, Pieridae,Lycaenidae, Nymphalidae, Danaidae, Satyridae, Hesperiidae, Sphingidae,Saturniidae, Geometridae, Arctiidae, Noctuidae, Lymantriidae, Sesiidae,and Tineidae.

Insect pests of the invention for the major crops include: Maize:Ostrinia nubilalis, European corn borer; Agrotis ipsilon, black cutworm;Helicoverpa zea, corn earworm; Spodoptera frugiperda, fall armyworm;Diatraea grandiosella, southwestern corn borer; Elasmopalpuslignosellus, lesser cornstalk borer; Diatraea saccharalis, sugarcaneborer; Diabrotica virgifera, western corn rootworm; Diabroticalongicornis barberi, northern corn rootworm; Diabrotica undecimpunctatahowardi, southern corn rootworm; Melanotus spp., wireworms; Cyclocephalaborealis, northern masked chafer (white grub); Cyclocephala immaculata,southern masked chafer (white grub); Popillia japonica, Japanese beetle;Chaetocnema pulicaria, corn flea beetle; Sphenophorus maidis, maizebillbug; Rhopalosiphum maidis, corn leaf aphid; Anuraphis maidiradicis,corn root aphid; Blissus leucopterus leucopterus, chinch bug; Melanoplusfemurrubrum, redlegged grasshopper; Melanoplus sanguinipes, migratorygrasshopper; Hylemya platura, seedcorn maggot; Agromyza parvicornis,corn blot leafminer; Anaphothrips obscrurus, grass thrips; Solenopsismilesta, thief ant; Tetranychus urticae, twospotted spider mite;Sorghum: Chilo partellus, sorghum borer; Spodoptera frugiperda, fallarmyworm; Helicoverpa zea, corn earworm; Elasmopalpus lignosellus,lesser cornstalk borer; Feltia subterranea, granulate cutworm;Phyllophaga crinita, white grub; Eleodes, Conoderus, and Aeolus spp.,wireworms; Oulema melanopus, cereal leaf beetle; Chaetocnema pulicaria,corn flea beetle; Sphenophorus maidis, maize billbug; Rhopalosiphummaidis; corn leaf aphid; Sipha flava, yellow sugarcane aphid; Blissusleucopterus leucopterus, chinch bug; Contarinia sorghicola, sorghummidge; Tetranychus cinnabarinus, carmine spider mite; Tetranychusurticae, twospotted spider mite; Wheat: Pseudaletia unipunctata, armyworm; Spodoptera frugiperda, fall armyworm; Elasmopalpus lignosellus,lesser cornstalk borer; Agrotis orthogonia, western cutworm;Elasmopalpus lignosellus, lesser cornstalk borer; Oulema melanopus,cereal leaf beetle; Hypera punctata, clover leaf weevil; Diabroticaundecimpunctata howardi, southern corn rootworm; Russian wheat aphid;Schizaphis graminum, greenbug; Macrosiphum avenae, English grain aphid;Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Melanoplus sanguinipes,migratory grasshopper; Mayetiola destructor, Hessian fly; Sitodiplosismosellana, wheat midge; Meromyza americana, wheat stem maggot; Hylemyacoarctata, wheat bulb fly; Frankliniella fusca, tobacco thrips; Cephuscinctus, wheat stem sawfly; Aceria tulipae, wheat curl mite; Sunflower:Suleima helianthana, sunflower bud moth; Homoeosoma electellum,sunflower moth; zygogramma exclamationis, sunflower beetle; Bothyrusgibbosus, carrot beetle; Neolasioptera murtfeldtiana, sunflower seedmidge; Cotton: Heliothis virescens, cotton budworm; Helicoverpa zea,cotton bollworm; Spodoptera exigua, beet armyworm; Pectinophoragossypiella, pink bollworm; Anthonomus grandis, boll weevil; Aphisgossypii, cotton aphid; Pseudatomoscelis seriatus, cotton fleahopper;Trialeurodes abutilonea, bandedwinged whitefly; Lygus lineolaris,tarnished plant bug; Melanoplus femurrubrum, redlegged grasshopper;Melanoplus differentialis, differential grasshopper; Thrips tabaci,onion thrips; Franklinkiella fusca, tobacco thrips; Tetranychuscinnabarinus, carmine spider mite; Tetranychus urticae, twospottedspider mite; Rice: Diatraea saccharalis, sugarcane borer; Spodopterafrugiperda, fall armyworm; Helicoverpa zea, corn earworm; Colaspisbrunnea, grape colaspis; Lissorhoptrus oryzophilus, rice water weevil;Sitophilus oryzae, rice weevil; Nephotettix nigropictus, riceleafhopper; Blissus leucopterus leucopterus, chinch bug; Acrosternumhilare, green stink bug; Soybean: Pseudoplusia includens, soybeanlooper; Anticarsia gemmatalis, velvetbean caterpillar; Plathypenascabra, green cloverworm; Ostrinia nubilalis, European corn borer;Agrotis ipsilon, black cutworm; Spodoptera exigua, beet armyworm;Heliothis virescens, cotton budworm; Helicoverpa zea, cotton bollworm;Epilachna varivestis, Mexican bean beetle; Myzus persicae, green peachaphid; Empoasca fabae, potato leafhopper; Acrosternum hilare, greenstink bug; Melanoplus femurrubrum, redlegged grasshopper; Melanoplusdifferentialis, differential grasshopper; Hylemya platura, seedcornmaggot; Sericothrips variabilis, soybean thrips; Thrips tabaci, onionthrips; Tetranychus turkestani, strawberry spider mite; Tetranychusurticae, twospotted spider mite; Barley: Ostrinia nubilalis, Europeancorn borer; Agrotis ipsilon, black cutworm; Schizaphis graminum,greenbug; Blissus leucopterus leucopterus, chinch bug; Acrosternumhilare, green stink bug; Euschistus servus, brown stink bug; Deliaplatura, seedcorn maggot; Mayetiola destructor, Hessian fly; Petrobialatens, brown wheat mite; Oil Seed Rape: Brevicoryne brassicae, cabbageaphid; Phyllotreta cruciferae, Flea beetle; Mamestra configurata, Berthaarmyworm; Plutella xylostella, Diamond-back moth; Delia ssp., Rootmaggots.

Nematodes include parasitic nematodes such as root-knot, cyst, andlesion nematodes, including Heterodera spp., Meloidogyne spp., andGlobodera spp.; particularly members of the cyst nematodes, including,but not limited to, Heterodera glycines (soybean cyst nematode);Heterodera schachtii (beet cyst nematode); Heterodera avenae (cerealcyst nematode); and Globodera rostochiensis and Globodera pailida(potato cyst nematodes). Lesion nematodes include Pratylenchus spp.

Methods for Increasing Plant Yield

Methods for increasing plant yield are provided. The methods compriseproviding a plant or plant cell expressing a polynucleotide encoding thepesticidal polypeptide sequence disclosed herein and growing the plantor a seed thereof in a field infested with a pest against which saidpolypeptide has pesticidal activity. In some embodiments, thepolypeptide has pesticidal activity against a lepidopteran, coleopteran,dipteran, hemipteran, or nematode pest, and said field is infested witha lepidopteran, hemipteran, coleopteran, dipteran, or nematode pest.

As defined herein, the “yield” of the plant refers to the quality and/orquantity of biomass produced by the plant. By “biomass” is intended anymeasured plant product. An increase in biomass production is anyimprovement in the yield of the measured plant product. Increasing plantyield has several commercial applications. For example, increasing plantleaf biomass may increase the yield of leafy vegetables for human oranimal consumption. Additionally, increasing leaf biomass can be used toincrease production of plant-derived pharmaceutical or industrialproducts. An increase in yield can comprise any statisticallysignificant increase including, but not limited to, at least a 1%increase, at least a 3% increase, at least a 5% increase, at least a 10%increase, at least a 20% increase, at least a 30%, at least a 50%, atleast a 70%, at least a 100% or a greater increase in yield compared toa plant not expressing the pesticidal sequence.

In specific methods, plant yield is increased as a result of improvedpest resistance of a plant expressing a pesticidal protein disclosedherein. Expression of the pesticidal protein results in a reducedability of a pest to infest or feed on the plant, thus improving plantyield.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1 Identification of a Protein active againstWestern Corn Rootworm from Strain ATX 2024

The Western Corn Rootworm active protein AXMI-205 was identified by acombination of biochemistry and genomics analysis from Strain ATX 2024.

ATX2024 was identified as an active strain in Diabrotica virgifera(Western Corn Rootworm or WCRW) bioassay exhibiting a heat sensitiveactivity. Protein fractionation and purification were performed onATX2024 culture materials as follows:

Cells of ATX2024 were grown in a suitable media (such as C2 media or CYSmedia supplemented with trehalose; the choice of media not beingcritical to the invention) for 3 days at 37° C. The incubation can alsobe performed at 30° C. Cell pellets were collected and cells disruptedin Buffer A (20 mM sodium acetate/50 mM sodium chloride, pH 5) using a“French Press” high pressure cell.

Lysates were clarified by centrifugation and dialyzed against 20 mMsodium acetate, 50 mM sodium chloride, pH 5.0. The dialyzed sample wasthen loaded onto a 20 ml SP Sepharose™ cation exchange column (GEHealthcare). Proteins were eluted with a linear salt gradient in BufferA from 50 mM to 1M sodium chloride over 20 column volumes. The elutioncan also be performed over 10 column volumes.

Active fractions were pooled and dialyzed against Buffer B (20 mMTris-HCl/50 mM NaCl, pH 7 or pH 8). Dialyzed active fractions were thenloaded on a 5 ml sepharose Q anion exchange column. Other anion exchangecolumns can be used, e.g., the 1.7 ml SOURCE™ Q anion exchange column.Proteins were eluted with a linear salt gradient in Buffer A from 50 mMto 1M NaCl. Collected fractions tested for activity on WCRW andfractions with activity on WCRW were observed. A protein band ofapproximately 52 kDa was identified as correlating with activity offractions. This protein is referred to herein as Protein Band #10.

Active fractions were then pooled and concentrated, and subjected toSDS-PAGE. The portion of the resulting gel corresponding to protein band#10 was isolated, and was submitted for analysis by both N-terminalsequencing and matrix-assisted laser desorption ionization tandemtime-of-flight mass spectrometry (MALDI-TOF-TOF) analyses as known inthe art.

Comparison of MALDI-TOF-TOF data from Protein Band #10 showed no matchesto a database of known proteins.

Amino acid sequencing of the N terminus of Protein Band #10 resulted inan N-terminal peptide sequence that showed no matches to known proteinsequences when compared to a database of known protein sequences.

Example 2 Genomic Sequencing of ATX2024

The complete gene sequence was identified from the selected strain asfollows:

Total DNA contains a mixture of some or all of the following: genomicDNA, plasmids of various size; phage chromosomes; other uncharacterizedextrachromosomal molecules.

Mechanical or enzymatic shearing of the extrachromosomal DNA to generatesize-distributed fragments.

Sequencing of the fragmented DNA by methods known in the art.

Example 3 Matching of N-Terminal and MALDI-TOF-TOF Data to GenomicSequence Data

A set of putative open reading frames (ORFs) encoded by the sequencedata for ATX2024 was generated by extracting all possible ORFs from thesequence reads generated from ATX2024. N-terminal sequencing data fromprotein band #10 (above) were compared to the set of ATX2024 ORFs usingthe BLAST algorithm. Two reads were found to encode putative proteinfragments with high homology to the N-terminal sequence data.

Similarly, MALDI-TOF-TOF data from protein band #10 were compared to theset of ATX2024 ORFs using the Mascot program (www.matrixscience.com;Perkins et al. (1999) Electrophoresis 20(18):3551-67). Seven reads werefound to encode putative protein fragments having significant matches topeaks present in the MALDI-TOF-TOF data set.

The DNA sequence reads identified from N-terminal and MALDI-TOF-TOF dataanalysis were assembled to provide a preliminary gene sequence.

TAIL-PCR strategies were used to obtain flanking sequence adjacent tothe preliminary gene sequence data. The sequences of the resulting PCRproducts were co-assembled with the original genomic data from ATX2024to provide a finished gene sequence for the open reading frame encodingProtein Band #10. This open reading frame is designated as Axmi205 (SEQID NO:1), and the protein encoded by the open reading frame as AXMI-205(SEQ ID NO:2, 3, or 4).

The genomic region encoding AXMI-205 was then amplified from the ATX2024genome, cloned, and the DNA sequence of this clone obtained. The DNAsequence of this clone in the region encompassing Axmi205 is provided asSEQ ID NO:12.

Comparison of AXMI-205 with known protein sequence databases shows thatAXMI-205 is a unique protein, showing very low homology (20% or less) toknown proteins.

Interestingly, AXMI-205 does show low, but possibly significant,homology to a broad class of loosely related proteins often referred toas MACPF proteins (Rosado et al, Cellular Microbiology (2008) 10(9),1765-1774). These proteins have been proposed to have roles in processessuch as immune response and protection from bacterial attack. AXMI-205is 20% identical to a protein from Clavibacter michiganensis (SEQ IDNO:14; GENBANK Accession No. YP_(—)001223127, Gartemann et al, J.Bacteriol. 190 (6), 2138-2149 (2008)) and 13% identity to a Photorhabdusluminescens protein (SEQ ID NO:15; GENBANK Accession No. 2QP2_A; Rosado,C. J., et al, Science 317 (5844), 1548-1551 (2007)). Although thesepercent identities are low, one can identify blocks of amino acidconservation between these proteins from inspection of FIG. 1.

Example 3 Heterologous Expression of AXMI-205

The open reading frame of Axmi205 was cloned into an E. coli expressionvector based on (1) maltose binding fusion vector to yield pAX6911, and(2) an expression vector based on pRSF1b to yield pAX7011.

For expression in E. coli, BL21*DE3 was transformed with either pAX6911,pAX7011, or control plasmids. A single colony transformed with thevector was inoculated in LB supplemented with kanamycin and grownovernight at 37° C. The following day, fresh medium was inoculated induplicate with 1% of overnight culture and grown at 37° C. tologarithmic phase. Subsequently, cultures were induced with 1 mM IPTGfor 3 hours at 37° C. or overnight at 20° C. Each cell pellet wassuspended in 50 mM sodium carbonate buffer, pH 10.5 supplemented with 1mM DTT and sonicated. Analysis by SDS-PAGE detected expression of aprotein corresponding to the predicted size of AXMI-205. In the case ofthe pMal fusion vector pAX6911, a protein consistent with the predictedsize for the pMAL-AXMI-205 fusion was observed by PAGE.

Example 4 Pesticidal Activity of AXMI-205

Fusion protein was purified from lysates of E. coli clones asrecommended by the supplier (New England Biolabs), and cleaved witheither Factor X_(a) or trypsin. Cleavage of the purified fusion proteinwas confirmed by SDS-PAGE analysis. Purified protein from pAX6911containing AXMI-205 and either pAX6911 cleaved with Factor Xa orTrypsin, or uncleaved protein were tested in insect assays withappropriate controls in a buffer composed of 20 mM Tris, 1 mM DTT, 50 mMNaCl. Soluble extracts of pAX7011 expressing AXMI-205 were also testedin this manner. After two days, samples containing AXMI-205 exhibitedstrong stunting activity and conferred mortality on Western CornRootworm. Table 1 shows a description of the scoring assignments usedherein, and Table 2 summarizes the activities observed from AXMI-205samples.

TABLE 1 Description of Scoring System Score Description 0 no effectobserved 1 mild non-uniform stunting 2 moderate non-uniform stunting 3moderate to severe uniform stunting 4 mortality (<100%) with uniformstunting 5 complete mortality

TABLE 2 Pesticidal Activity of AXMI-205 samples. WCRW Activity PercentSample (2 days) Mortality Axmi205 MBP fusion (from pAX6911) 3.0 25%Axmi205 MBP fusion cleaved with Factor Xa 3.0 25% Axmi205 MBP fusioncleaved with trypsin 3.0 25% Axmi205 in soluble extract from pAX7011 3.00% Buffer Control 0 0%

Example 5 Additional Assays for Pesticidal Activity

The nucleotide sequences of the invention can be tested for theirability to produce pesticidal proteins. The ability of a pesticidalprotein to act as a pesticide upon a pest is often assessed in a numberof ways. One way well known in the art is to perform a feeding assay. Insuch a feeding assay, one exposes the pest to a sample containing eithercompounds to be tested or control samples. Often this is performed byplacing the material to be tested, or a suitable dilution of suchmaterial, onto a material that the pest will ingest, such as anartificial diet. The material to be tested may be composed of a liquid,solid, or slurry. The material to be tested may be placed upon thesurface and then allowed to dry. Alternatively, the material to betested may be mixed with a molten artificial diet, then dispensed intothe assay chamber. The assay chamber may be, for example, a cup, a dish,or a well of a microtiter plate.

Assays for sucking pests (for example aphids) may involve separating thetest material from the insect by a partition, ideally a portion that canbe pierced by the sucking mouth parts of the sucking insect, to allowingestion of the test material. Often the test material is mixed with afeeding stimulant, such as sucrose, to promote ingestion of the testcompound.

Other types of assays can include microinjection of the test materialinto the mouth, or gut of the pest, as well as development of transgenicplants, followed by test of the ability of the pest to feed upon thetransgenic plant. Plant testing may involve isolation of the plant partsnormally consumed, for example, small cages attached to a leaf, orisolation of entire plants in cages containing insects.

Other methods and approaches to assay pests are known in the art, andcan be found, for example in Robertson and Preisler, eds. (1992)Pesticide bioassays with arthropods, CRC, Boca Raton, Fla.Alternatively, assays are commonly described in the journals ArthropodManagement Tests and Journal of Economic Entomology or by discussionwith members of the Entomological Society of America (ESA).

Example 6 Synthetic Genes

Synthetic genes encoding AXMI-205 were designed. Axmi205v01.02 is setforth in SEQ ID NO:9. Axmi205v01.03 is set forth in SEQ ID NO:10.Axmi205v01.04 is set forth in SEQ ID NO:11.

Example 7 Variants of AXMI-205

To identify regions and positions in the C-terminal portion of AXMI-205that are functionally important, alanine scanning mutants were assayedin the region corresponding to amino acid positions 307-536 of SEQ IDNO:2. The alanine mutants were generated synthetically (Geneart,Burlingame, Calif.) and were organized in an expression vector derivedfrom pAX3577 for expression in E. coli (pAX3577 contains Axmi250v01.03in pRSF1b(Invitrogen)).

Starting with mutant 5307A, every second residue was substituted with analanine. The last alanine mutant in this series was K535A. In total, 101alanine mutants were pooled.

The pooled alanine mutants, as well as pAX3577, were transformed intoBL21*DE3 cells and plated on LB+Kanamycin (100 μg/ml). Fresh colonieswere picked into 8 ml LB+Kanamycin (100 μg/ml) liquid medium and weregrown in 24 deep well blocks at 37° C. and 300 rpm until an OD600 nm of0.6 was reached. IPTG was added to a final concentration of 0.5 mM andthe cultures were incubated for an additional 18 hours at 20° C. TheOD600 nm was determined and the cells were collected by centrifugation(10 minutes at 4000 rpm, 4° C.). The cell pellets were resuspended in 20mM Tris/HCl pH7.4, 150 mM NaCl, 1 mM DTT at a density of 20 OD600/ml.The cells were disrupted by bead beating and soluble extracts wereobtained after centrifugation at 4500 rpm for 15 minutes at 4° C.

The extracts were assayed for activity against WCRW at four replicatesper variant each. After five and six days, rootworm toxicity scores weredetermined by averaging the scores from the four replicates. 266variants were screened in this primary screen, providing a 3-foldcoverage of the library. Variants scoring above and below the score ofthe Axmi205 wild-type sequence were sequenced.

The following alanine mutants (relative to SEQ ID NO:2) were found to beactive on WCRW: 5307A, D315A, V317A, S349A, G351A, K353A, V355A, D395A,G399A, W407A, G419A, P355A, P435A, S443A, K465A, V467A, F483A, P487A,S495A, D497A, E499A, K509A, and 1513A. The alanine mutant E499A wasdesignated Axmi205(evo24) (SEQ ID NO:5) and the alanine mutant V467A wasdesignated Axmi205(evo25) (SEQ ID NO:6).

Example 8 Activity of Truncations of Axmi-205

Several truncations of axmi-205 were constructed and tested for activityon western corn rootworm. C-terminal truncations were constructed thatremoved either 10, 20, 30, 34, or 71 amino acids from the C-terminus ofthe AXMI-205 protein (SEQ ID NO:2).

Clone pAX7106 expressed an MBP fusion that, after cleavage with factorXa, produced the protein AXMI-205(trunc10) (SEQ ID NO:7), which islacking 10 amino acids from the C-terminus relative to AXMI-205. ClonepAX7106 expressed an MBP fusion protein that, after cleavage with factorXa, produced the protein AXMI-205(trunc20) (SEQ ID NO:8), which islacking 20 amino acids from the C-terminus relative to AXMI-205. BothAXMI-205(trunc10) and AXMI-205(trunc20) demonstrated activity on WCRW,whereas a truncation of 30 amino acids did not.

Example 9 Vectoring of Genes for Plant Expression

The coding regions of the invention are connected with appropriatepromoter and terminator sequences for expression in plants. Suchsequences are well known in the art and may include the rice actinpromoter or maize ubiquitin promoter for expression in monocots, theArabidopsis UBQ3 promoter or CaMV 35S promoter for expression in dicots,and the nos or PinII terminators. Techniques for producing andconfirming promoter—gene—terminator constructs also are well known inthe art.

In one aspect of the invention, synthetic DNA sequences are designed andgenerated. These synthetic sequences have altered nucleotide sequencerelative to the parent sequence, but encode proteins that areessentially identical to the parent AXMI-205 protein (e.g., SEQ IDNO:9-12).

In another aspect of the invention, modified versions of the syntheticgenes are designed such that the resulting peptide is targeted to aplant organelle, such as the endoplasmic reticulum or the apoplast.Peptide sequences known to result in targeting of fusion proteins toplant organelles are known in the art. For example, the N-terminalregion of the acid phosphatase gene from the White Lupin Lupinus albus(GENBANK® ID GI:14276838, Miller et al. (2001) Plant Physiology 127:594-606) is known in the art to result in endoplasmic reticulumtargeting of heterologous proteins. If the resulting fusion protein alsocontains an endoplasmic reticulum retention sequence comprising thepeptide N-terminus-lysine-aspartic acid-glutamic acid-leucine (i.e., the“KDEL” motif, SEQ ID NO:13) at the C-terminus, the fusion protein willbe targeted to the endoplasmic reticulum. If the fusion protein lacks anendoplasmic reticulum targeting sequence at the C-terminus, the proteinwill be targeted to the endoplasmic reticulum, but will ultimately besequestered in the apoplast.

Thus, this gene encodes a fusion protein that contains the N-terminalthirty-one amino acids of the acid phosphatase gene from the White LupinLupinus albus (GENBANK® ID GI:14276838, Miller et al., 2001, supra)fused to the N-terminus of the amino acid sequence of the invention, aswell as the KDEL sequence at the C-terminus. Thus, the resulting proteinis predicted to be targeted the plant endoplasmic reticulum uponexpression in a plant cell.

The plant expression cassettes described above are combined with anappropriate plant selectable marker to aid in the selection oftransformed cells and tissues, and ligated into plant transformationvectors. These may include binary vectors from Agrobacterium-mediatedtransformation or simple plasmid vectors for aerosol or biolistictransformation.

Example 10 Vectoring Genes for Plant Expression

The coding region DNA of the genes of the invention are operablyconnected with appropriate promoter and terminator sequences forexpression in plants. Such sequences are well known in the art and mayinclude the rice actin promoter or maize ubiquitin promoter forexpression in monocots, the Arabidopsis UBQ3 promoter or CaMV 35Spromoter for expression in dicots, and the nos or PinII terminators.Techniques for producing and confirming promoter—gene—terminatorconstructs also are well known in the art.

The plant expression cassettes described above are combined with anappropriate plant selectable marker to aid in the selections oftransformed cells and tissues, and ligated into plant transformationvectors. These may include binary vectors from Agrobacterium-mediatedtransformation or simple plasmid vectors for aerosol or biolistictransformation.

Example 11 Transformation of Maize Cells with the Pesticidal ProteinGenes Described Herein

Maize ears are best collected 8-12 days after pollination. Embryos areisolated from the ears, and those embryos 0.8-1.5 mm in size arepreferred for use in transformation. Embryos are plated scutellumside-up on a suitable incubation media, such as DN62A5S media (3.98 g/LN6 Salts; 1 mL/L (of 1000× Stock) N6 Vitamins; 800 mg/L L-Asparagine;100 mg/L Myo-inositol; 1.4 g/L L-Proline; 100 mg/L Casamino acids; 50g/L sucrose; 1 mL/L (of 1 mg/mL Stock) 2,4-D). However, media and saltsother than DN62A5S are suitable and are known in the art. Embryos areincubated overnight at 25° C. in the dark. However, it is not necessaryper se to incubate the embryos overnight.

The resulting explants are transferred to mesh squares (30-40 perplate), transferred onto osmotic media for about 30-45 minutes, thentransferred to a beaming plate (see, for example, PCT Publication No.WO/0138514 and U.S. Pat. No. 5,240,842).

DNA constructs designed to the genes of the invention in plant cells areaccelerated into plant tissue using an aerosol beam accelerator, usingconditions essentially as described in PCT Publication No. WO/0138514.After beaming, embryos are incubated for about 30 min on osmotic media,and placed onto incubation media overnight at 25° C. in the dark. Toavoid unduly damaging beamed explants, they are incubated for at least24 hours prior to transfer to recovery media. Embryos are then spreadonto recovery period media, for about 5 days, 25° C. in the dark, thentransferred to a selection media. Explants are incubated in selectionmedia for up to eight weeks, depending on the nature and characteristicsof the particular selection utilized. After the selection period, theresulting callus is transferred to embryo maturation media, until theformation of mature somatic embryos is observed. The resulting maturesomatic embryos are then placed under low light, and the process ofregeneration is initiated by methods known in the art. The resultingshoots are allowed to root on rooting media, and the resulting plantsare transferred to nursery pots and propagated as transgenic plants.

Materials

DN62A5S Media

Components Per Liter Source Chu's N6 Basal 3.98 g/L Phytotechnology SaltMixture Labs (Prod. No. C 416) Chu's N6 Vitamin   1 mL/L (of 1000xStock) Phytotechnology Solution Labs (Prod. No. C 149) L-Asparagine  800mg/L Phytotechnology Labs Myo-inositol  100 mg/L Sigma L-Proline  1.4g/L Phytotechnology Labs Casamino acids  100 mg/L Fisher ScientificSucrose   50 g/L Phytotechnology Labs 2,4-D (Prod.)   1 mL/L (of 1 mg/mLStock) Sigma No. D-7299)

The pH of the solution is adjusted to pH 5.8 with 1N KOH/1N KCl, Gelrite(Sigma) is added at a concentration up to 3 g/L, and the media isautoclaved. After cooling to 50° C., 2 ml/L of a 5 mg/ml stock solutionof silver nitrate (Phytotechnology Labs) is added.

Example 12 Transformation of Genes of the Invention in Plant Cells byAgrobacterium-Mediated Transformation

Ears are best collected 8-12 days after pollination. Embryos areisolated from the ears, and those embryos 0.8-1.5 mm in size arepreferred for use in transformation. Embryos are plated scutellumside-up on a suitable incubation media, and incubated overnight at 25°C. in the dark. However, it is not necessary per se to incubate theembryos overnight. Embryos are contacted with an Agrobacterium straincontaining the appropriate vectors for Ti plasmid mediated transfer forabout 5-10 min, and then plated onto co-cultivation media for about 3days (25° C. in the dark). After co-cultivation, explants aretransferred to recovery period media for about five days (at 25° C. inthe dark). Explants are incubated in selection media for up to eightweeks, depending on the nature and characteristics of the particularselection utilized. After the selection period, the resulting callus istransferred to embryo maturation media, until the formation of maturesomatic embryos is observed. The resulting mature somatic embryos arethen placed under low light, and the process of regeneration isinitiated as known in the art.

Example 13 Protection of Transgenic Plants Expressing Axmi205 from RootDamage Upon Infestation with Western Corn Rootworm

Transgenic maize plants transformed with either of two versions ofAxmi205 (Axmi205 (SEQ ID NO:1) or Axmi205v01.03 (SEQ ID NO:10)) wereobtained by Agrobacterium-mediated transformation. Plants shown by PCRanalysis to contain the appropriate Axmi205 construct were selected, andtransferred to root trainer containers.

T₀ plants containing Axmi205 or Axmi205v01.03 were transplanted toroot-trainer containers and propagated for approximately three weeks.Individual plants were then each infested with ˜125 non-diapausingwestern corn rootworm (Diabrotica virgifera) eggs. Greater than 90% ofthe eggs were observed to have hatched within 24 hours of infestation.Plants were analyzed for expression of AXMI-205 protein by Western blotanalysis using an anti-AXMI-205 antibody. Plants expressing detectableamounts of AXMI-205 were selected for analysis. After fifteen days, theamount of root damage in each plant was assessed using the Iowa Statenode injury scale 1 (Oleson, J. D., Y. Park, T. M. Nowatzki, and J. J.Tollefson. 2005. J. Econ Entomol. 98(1): 1-8). Table 3 shows that bothforms of AXMI-205 resulted in lower root damage than control plantsinfested in the same manner. In similar experiments, plants containingeither Axmi205v01.02 or Axmi-205v01.04 demonstrated improved rootratings compared to the nontransformed controls (not shown).

TABLE 3 Root damage from transgenic maize expressing Axmi-205 Number ofAverage Root Transgene Plants Score Variance Control plants (notransgene) 35 2.44 0.23 Axmi205 16 1.11 0.5 Axmi205v01.03 12 0.81 0.43

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

That which is claimed:
 1. A method for isolating or recovering from asample containing nucleic acid sequences a nucleotide sequence encodinga polypeptide with a pesticidal activity comprising: (a) providing apolynucleotide probe comprising the complement of the nucleic acidsequence of any of SEQ ID NO: 1, 9, 10, 11 or 12, or a fragment thereofcomprising at least 500 consecutive nucleotides of SEQ ID NO: 1, 9, 10,11 or 12; (b) contacting the sample with the polynucleotide probe understringent hybridization conditions, wherein said conditions comprisehybridization for 4 to 12 hours in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C.; (c) isolating orrecovering from said sample a nucleic acid that specifically hybridizeswith the polynucleotide probe of (a); and (d) assaying the polypeptideencoded by the nucleic acid of step (c) for pesticidal activity.